Recombinant expression of the rat vesicular acetylcholine transporter

ABSTRACT

A nucleic sequence coding for a protein involved in the vesicular transport of acetylcholine, the corresponding protein and the promoter sequences implicated in expressing said protein are disclosed. The invention also discloses expression vectors containing said sequence and the therapeutic use of said sequence or said vectors.

This application was filed under 35 USC 371 as a national stage ofInternational Application PCT/FR95/01073, filed Aug. 10, 1995, and whichdesignated the United States.

The present invention relates to a nucleic acid sequence coding for aprotein involved in the vesicular transport of acetylcholine and to thecorresponding protein. It also relates to expression vectors integratingthe said sequence and to the use of this sequence or the said vectorsfor therapeutic purposes.

Acetylcholine, ACh, is a neurotransmitter synthesized by the enzymecholine acetyltransferase, ChAT. At the ends of the cholinergic neurons,the majority of the ACh produced is transported from the cytoplasm tothe inside of the synaptic vesicles. The accumulation of ACh in thesevesicles is mediated by the activity of an ATPase which pumps H⁺ ions,generating an electrochemical gradient (Anderson D. C. et al., (1982)Biochemistry 21, 3037-3043). This gradient is utilized by a transporterto take up ACh via an exchange of protons (Parsons S. M. et al., (1993),Int. Rev. neurobiol. 35, 279-390). This type of mechanism is comparableto the one involved in the transport of biogenic amines into thesynaptic vesicles.

An understanding of the different mechanisms of regulation participatingin the expression of ACh would be especially valuable from a therapeuticstandpoint.

Recently, cDNAs coding for vesicular ACh transporters in Caenohabditiselegans and Torpedo have been cloned and sequenced (Alfonso A. et al.,(1993) Science 261, 617-619; Varoqui H. et al., FEBS Letters 342,97-102). A study of the sequences of the corresponding proteins revealedthe existence of structural similarities between these two proteins, andlikewise with respect to two vesicular transporters of biogenic aminesin mammals, VMAT1 and VMAT2 (Liu et al., (1992) Cell 70, 539-551). Thesecommon structural features are, in particular, (i) 12 transmembranedomains (TM), (ii) the presence in these transmembrane domains ofcharged amino acid residues which probably participate in the transportof substrate, (iii) a glycosylated loop localized between TM1 and TM2,and (iv) cytoplasmic C- and N-terminal ends which display fewersimilarities than the remainder of the protein.

The subject of the present invention is, more especially, the isolation,sequencing and characterization of a region of the gene coding for ChATcapable of expressing a vesicular ACh transporter, as well as theidentification of promoter sequences involved in the expression of thistransporter. It also describes cassettes for expression of this gene,vectors containing it and their use for directing the expression of thistransporter.

More specifically, the present invention relates to a nucleic acidsequence coding for a protein involved in the vesicular transport ofACh, characterized in that it is localized within the first intron ofthe gene coding for ChAT, in the same transcriptional orientation.

From the 5′ region of the gene coding for rat ChAT, and more preciselyfrom a restriction map of this region, the inventors isolated aHindIII-BamHI fragment carrying the first intron of this gene and, inpart, the sequences of the first two corresponding R and N exons,respectively, at the 5′ and 3′ ends of this fragment. Unexpectedly, thecloning and sequencing of this fragment revealed the presence of a1590-bp open reading frame coding for a protein of the order of 530amino acids, equivalent to a mass of the order of 56.5 kDa, anddisplaying similarities in respect of its sequence with proteins of thetransporter type. This protein was identified as a rat vesicular AChtransporter and is designated hereinafter rVAT.

This DNA sequence is, more specifically, localized within the firstintron of the gene coding for ChAT, downstream of the R-type promoter ofthe ChAT gene and in the same transcription orientation as the ChATgene. It is not interrupted by any intron.

More especially, the present invention relates to a nucleic acidsequence coding for a vesicular ACh transporter, characterized in thatit comprises all or part of SEQ ID No. 1 or one of its derivatives.

Preferably, the sequence comprises all or part of the sequence SEQ IDNo.2 or one of its derivatives.

For the purposes of the present invention, the term derivative denotesany sequence differing from the sequence under consideration as a resultof the degeneracy of the genetic code, obtained by one or moremodifications of a genetic and/or chemical nature, as well as anysequence hybridizing with these sequences or fragments of the latter andwhose product possesses the stated activity. Modification of a geneticand/or chemical nature may be understood to mean any mutation,substitution, deletion, addition and/or modification of one or moreresidues. Such derivatives may be generated for different purposes, suchas, in particular, that of increasing the affinity-of the correspondingpolypeptide for its ligand(s), that of improving its levels ofproduction, that of increasing its resistance to proteases, that ofincreasing and/or modifying its activity or that of endowing it with newpharmacokinetic and/or biological properties. Among derivativesresulting from an addition, chimeric nucleic acid sequences containingan additional heterologous portion linked to one end may, for example,be mentioned. The term derivative also comprises sequences homologous tothe sequence under consideration, originating from other cellularsources, and in particular from cells of human origin, or from otherorganisms, and possessing an activity of the same type. Such homologoussequences may be obtained by hybridization experiments. Thehybridizations may be carried out using nucleic acid libraries,employing as probe the native sequence or a fragment of the latter,under conventional stringency conditions (Maniatis et al., see generaltechniques of molecular biology), or preferably under high stringencyconditions.

The present invention shall also be understood to cover thecorresponding antisense sequences whose expression enables transcriptionof cellular mRNAs to be controlled. Such sequences can consist of all orpart of the nucleic acid sequence under consideration, transcribed inthe reverse orientation.

More preferably, the sequence in question is the nucleic acid sequencecoding for the rat vesicular ACh transporter, rVAT.

Bearing in mind the localization of the gene according to the invention,namely in the first intron of the gene coding for ChAT, downstream ofthe R-type ChAT promoter and in the same transcriptional orientation,the inventors sought to find out whether the ChAT and VAT mRNAs could beexpressed from the same promoter.

This hypothesis was verified experimentally. Two forms of mRNA codingfor the VAT transporter were identified. They contain all or part of thesequence of the R1- or R2-type R exon (of ChAT mRNA) (Kengaku et al.Mol. Brain Res. 18: 71-76 (1993)) (FIG. 1), immediately followed by thesequence of a second exon beginning 308 bp upstream of the translationinitiation codon (of VAT). Besides these two mRNAs, there are threeother forms of VAT mRNA which are apparently more abundant than the twoforms mentioned above, coding for the polypeptide which is the subjectof the invention. One form of VAT mRNA contains all or part of the Rexon mentioned above. The 5′ ends of the other two forms are localizeddownstream of the R exon. The form which we designate V1, ofapproximately 2.6 kb, has two 5′ ends located 426 bp and 402 bp upstreamof the translation initiation codon of VAT (positions 949 and 972,respectively, on SEQ ID No. 1). The form of VAT mRNA which we designateV2, of approximately 3 kb, has several 5′ ends located between 863 bpand 888 bp upstream of the translation initiation codon of VAT(positions 486 to 511 of SEQ ID No. 1).

The subject of the present invention is also promoter regions involvedin the expression of VAT and localized in the gene coding for ChAT. Suchregions are, more especially, the promoter region comprising all or partof SEQ ID No.4 or one of its derivatives, and attached promoter regionslocalized in SEQ ID No. 1.

For the purposes of the present invention, promoter region denotes thesequence or sequences responsible for the expression of VAT. Thisapplies especially to promoter, activation and regulatory sequencesand/or sequences permitting a tissue-specific expression.

SEQ ID No.4 is already known to control the expression of the ChAT gene(Bejanin et al., J. Neurochem.58: 1580-1583 (1992)). Unexpectedly, thisregion proved also to be responsible for the expression of the genecoding for VAT, and more specifically for at least two types of VATmRNA. It was thus demonstrated that VAT and ChAT mRNAs may be producedfrom the same R-type ChAT promoter. Furthermore, two promoter regionsresponsible for the production of V1- and V2-type VAT mRNAs have beenidentified downstream of the R exon in the first intron of the ChATgene. V1-type VAT mRNA is produced from a promoter located betweenpositions 584 and 1027 of SEQ ID No. 1, whereas V2-type mRNA is producedfrom a promoter lying between positions 2 and 583 of the sequence SEQ-IDNo. 1.

The subject of the present invention is also the use of these promoterregions-to control and/or participate in the expression of genes. Thesepromoter regions are also advantageous for targeting the expression of aprotein in cholinergic neurons. Naturally, these promoter regions areespecially useful for directing the expression of a vesicularacetylcholine transport protein, the expression being coupled, whereappropriate, with that of another gene.

The invention relates, in addition, to a polypeptide involved in thevesicular transport of ACh, capable of being expressed by a nucleic acidsequence as described above.

The polypeptides of the invention may be obtained by expression in acell host of a nucleotide sequence as described above, by chemicalsynthesis, on the basis of the sequence SEQ ID No. 2 using techniquesknown to a person skilled in the art, or by a combination of thesetechniques.

Comparison of this protein SEQ ID No. 3 with proteins already known asan ACh transporter, such as those of Torpedo or Caenorhabditis elegans,has enabled some similarities to be demonstrated. Thus, the proteinaccording to the invention possesses approximately 77% homology with theTorpedo protein and 56% homology with the Caenorhabditis elegansprotein, out of 352 amino acids.

In the case of the abovementioned significant homologies, it is notedespecially that the 12 transmembrane domains, (TM), representing thealready known transporters, exist in the protein according to theinvention. In particular, the aspartic acid residues of transmembranedomains 1, 6, 10 and 11 and the lysine residue of transmembrane domain2, which probably participate in the binding with the substrate, arepresent. The considerable disparity between the protein according to theinvention and other known transporters occurs in the highly hydrophilicloop located between the first two transmembrane domains and the N- andC-terminal ends. In the case of the present invention, the loopintegrates two potential N-glycosylation sites.

These observations collectively demonstrate that the claimed proteinbelongs to the family of vesicular neurotransmitter transporterscontaining 12 transmembrane domains.

More specifically, it is a protein which comprises all or part of SEQ IDNO: 3 or a derivative thereof. For the definition of the termderivative, reference may be made to the definition put forward above.

More preferably, it is the rat vesicular ACh transporter, hereinafterdesignated rVAT.

Preferably, the nucleic acid sequences according to the invention formpart of a vector. The use of such a vector makes it possible, in effect,to improve the administration of the nucleic acid in the cells to betreated, and also to increase its stability in the said cells, therebyenabling a lasting therapeutic effect to be obtained.

The vector used may be of various origins, provided it is capable oftransforming animal cells, preferably human nerve cells. In a preferredembodiment of the invention, a viral vector is used, which may be chosenfrom adenoviruses, retroviruses, adeno-associated viruses (AAV),herpesvirus, cytomegalovirus (CMV), vaccinia virus, and the like.Vectors derived from adenoviruses, from retroviruses or from AAVincorporating heterologous nucleic acid sequences have been described inthe literature [Akli et al., Nature Genetics 3 (1993) 224;Stratford-Perricaudet et al., Human Gene Therapy 1 (1990) 241; EP 185573, Levrero et al., Gene 101 (1991) 195; Le Gal la Salle et al.,Science 259 (1993) 988; Roemer and Friedmann, Eur. J. Biochem. 208(1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., NewBiol. 2 (1990) 739; Miyanohara et al., New Biol. 4 (1992) 238;WO91/18088].

Hence the present invention also relates to any recombinant viruscomprising, inserted into its genome, a nucleic acid sequence as definedabove.

Advantageously, the recombinant virus according to the invention is adefective virus. The term “defective virus” denotes a virus incapable ofreplication in the target cell. Generally, the genome of the defectiveviruses used in the context of the present invention hence lacks atleast the sequences needed for replication of the said virus in theinfected cell. These regions may be either removed (wholly orpartially), or rendered non-functional, or substituted by othersequences, and in particular by the nucleic acid of the invention.Preferably, the defective virus nevertheless retains the sequences ofits genome which are needed for encapsidation of the viral particles.

It is especially advantageous to use the nucleic acid sequences of theinvention in a form incorporated in a defective recombinant adenovirus,AAV or retrovirus.

As regards adenoviruses, there are different serotypes of these, whosestructure and properties vary somewhat but which are not pathogenic forman, and in particular non-immunosuppressed subjects. Moreover, theseviruses do not integrate in the genome of the cells they infect, and canincorporate large fragments of exogenous DNA. Among the differentserotypes, it is preferable to use, in the context of the presentinvention, adenoviruses type 2 or 5 (Ad 2 or Ad 5). In the case of Ad 5adenoviruses, the sequences needed for replication are the E1A and E1Bregions.

The defective recombinant viruses of the invention may be prepared byhomologous recombination between a defective virus and a plasmidcarrying, inter alia, the nucleotide sequence as defined above (Levreroet al., Gene 101 (1991) 195; Graham, EMBO J. 3(12) (1984) 2917).Homologous recombination takes place after cotransfection of the saidvirus and said plasmid in a suitable cell line. The cell line usedshould preferably (i) be transformable by the said elements, and (ii)contain the sequences capable of complementing the portion of the genomeof the defective virus, preferably in integrated form in order to avoidrisks of recombination. As an example of a line which can be used forthe preparation of defective recombinant adenoviruses, there may bementioned the human embryonic kidney line 293 (Graham et al., J. Gen.Virol. 36 (1977) 59) which contains, in particular, integrated in itsgenome, the left-hand portion of the genome of an Ad5 adenovirus (12%).As an example of a line which can be used for the preparation ofdefective recombinant retroviruses, the CRIP line (Danos and Mulligan,PNAS 85 (1988) 6460) may be mentioned.

Thereafter, the viruses which have multiplied are recovered and purifiedaccording to standard techniques of molecular biology.

The subject of the present invention is also a pharmaceuticalcomposition comprising at least one nucleotide sequence, vector orpolypeptide according to the invention.

It also relates to any use of a sequence or vector as are claimed forthe preparation of a pharmaceutical composition intended for thetreatment of pathologies affecting the nervous system.

The nucleic acid sequence, as claimed, coding for an acetylcholinetransporter is, in addition, most especially useful for screening newbiologically active products, and especially those involved in theexpression and/or regulation of-acetylcholine.

The subject of the present invention is also transgenic animals into thegenome of which at least one nucleic acid sequence, as claimed, codingfor an acetylcholine transporter is inserted.

The examples and figures presented below by way of illustration andwithout implied limitation bring out other advantages and features ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Diagrammatic representation of the 5′ region of the rat ChATgene and of the different ChAT mRNAs The clear and black boxes indicate,respectively, the coding and non-coding exons. R, N and M are the 3non-coding ChAT exons. The 1590-bp open reading frame identified fromthe sequencing of a 3940-bp HindIII(H)-BamHI(B) gene fragment, is alsoshown. The potential translation initiation codon, ATG (position +1), isflanked by a cytosine (position −1) and two guanosine residues(positions −3 and +4), which tally with the consensus sequence of Kozak(Kozak M. J., Cell. Biol. 115, 887-903 1991). It is preceded by a stopcodon in frame located 726 bp upstream. The open reading frame isfollowed by consensus polyadenylation sequences. The most probable one,designated PA (AATAAA) (SEQ ID No. 5), is located 389 bp downstream ofthe termination codon.

FIG. 2: Analysis by the Northern technique of the distribution of rVATmRNAs in different rat tissues with a probe specific for the openreading frame (position 31-1724 as defined in FIG. 1)

Lane 1: Spinal cord poly(A)⁺ RNA (1 μg);

Lanes 2-11: 10 μg of:

2,3: spinal cord RNA (2 different preparations);

4: brain stem RNA;

5: adrenal gland poly(A)⁺ RNA;

6: olfactory bulb RNA;

7: cerebellum RNA;

8: liver RNA;

9: spinal cord RNA;

10: septal region poly(A)⁺ RNA;

11: striatum poly(A)⁺ RNA.

The RNA blots are prepared in the presence of RNA molecular weightmarkers (Gibco BRL). Lanes 1 to 8 and 9 to 11 represent the results oftwo independent experiments. The autoradiographs are exposed for threedays at a temperature of −70° C. with an intensifying screen.

FIG. 3: Diversity of the rVAT mRNAs in rat spinal cord

The figure shows the results of Southern blot analysis of the cDNAamplification products (lanes 1,2) and of the SLIC products (lanes 3,4).The clear and black boxes indicate, respectively, the non-coding andcoding exons. The oligonucleotides used for the synthesis of cDNA (P),for the amplification (two sense primers 1,2 upstream and a commonantisense primer L) and for the Southern hybridization (H) are indicateddiagrammatically by arrows. The poly(A)⁺ RNA is subjected to a synthesisof the first strand of cDNA with or, as a control, without reversetranscriptase. The amplifications of the cDNA (+) or the controls (−)with the pair of oligonucleotides L and 1 or 2 are shown, respectively,in lanes 1 and 2. The amplifications with oligonucleotides L and A5′l ofthe CDNA (+) or the controls (−), ligated or otherwise, are seen,respectively, in lanes 3 and 4. The vertical bars in the exons R andrVAT represent the splicing sites deduced from the sequences of the twoDNA fragments obtained in lane 1. The 5′ splicing site in the R exoncorresponds to the one shown in FIG. 1. The consensus sequence of the 3′splicing site is underlined. The position of the nucleotide locatednearest the 5′ end for the SLIC products sequenced is indicated by anasterisk.

EXAMPLES Example 1

Synthesis of the First Strand of cDNA (primer extension) and Ligation ofan Oligonucleotide to the Single-stranded cDNA (SLIC)

The RNA is extracted from rat spinal cord using the RNAzol method(Bioprobe system). The poly(A)⁺ RNA is purified using Dynabeads® kit(Dynal). The first strand of cDNA is synthesized from 1 μg of poly(A)⁺RNA in a reverse transcription buffer in the presence of 100 μg/ml BSA,15 units of RNasin (Promega), 14 mM β-mercaptoethanol, 1 mM dNTPs, 4 mMpyrophosphate, 7.5 pM primer oligonucleotide P (FIG. 3) and 40 units ofreverse transcriptase (RTase, Promega). The reaction mixture isincubated for 45 minutes at 42° C. A control is performed in the absenceof RTAse. cDNA and controls are heated for 5 minutes at 95° C. in orderto denature the DNA-RNA heteroduplexes, and then purified with thePrep-A-Gene® Kit (Biorad). In the SLIC experiments, a 50-meroligonucleotide (A5′NV) is ligated to the 3′ end of the first strand ofpurified cDNA using T4 RNA ligase (Boehringer) as described in theliterature (Dumas et al., Nucleic Acids Res. 19 5227-5232, 1991). Acontrol is performed in the absence of the enzyme. The ligation productsand controls are then purified using the Prep-A-Gene® Kit.

Example 2

Amplification Experiments

The cDNAs and the SLIC products are amplified in a Techne ThermalCycler® (30 or 40 amplification cycles, respectively). Each cycleconsists of: 45s at 94° C., 45s at 65° C. and 50s at 72° C. The senseprimers used are the following: oligonucleotides 1 or 2 for the cDNAsand the oligonucleotide A5′1 (complementary to a portion of A5′NV) forthe cDNAs ligated at the 3′ end. A common antisense primer L is used.The amplification products are separated on 2% agarose gel and analysedby the Southern blotting technique using the oligonucleotide H as probe.The portions of the gel containing the bands observed are isolated, andthe DNA they contain is purified, subdloned into plasmid pUC19(Appligene) and sequenced on both strands by means of the Sequenase® kit(USB, Amersham). The sequences and the positions (see FIG. 1) of theoligonucleotides used are:

1 (−1486, 5′-CTGTCGCTGCAAGCCAGGACTCT-3′) (SEQ ID No. 6)

2 (−410, 5′-TGGAGGAAGAGGCAAGAGCGGA-3′) (SEQ ID No. 7)

H (+24, 5′-ACCGGTTGGCGCGGTGGGTTCCAT-3′) (SEQ ID No. 8)

L (+62, 5′-CACCGCTTCCGACAGTTTGGTG-3′) (SEQ ID No. 9)

P (+187, 5′-TGGGCGATATAGTCGGGAACAATG-3′) (SEQ ID No. 10)

A5′NV (5′-CTGCATCTATCTAATGCTCCTCTCGCTACCTGCTCACTCTGCGTGACATC-3′) (SEQ IDNo. 11)

A5′1 (5′-GATGTCACGCAGAGTGAGCAGGTAG-3′) (SEQ ID No. 12)

Example 3

Analysis of the RNA

The RNAs or poly(A)⁺ RNAs are prepared from rat tissues as describedabove, fractionated on agarose gel (1%)/formaldehyde and transferredonto nylon membranes (Hybond N⁺, Amersham) as described in theliterature (Faucon Biguet et al., EMBO J., 5 287-291 1986). The filtersare hybridized at 42° C., in a buffer containing 50% (vol/vol) offormamide, with an. SmaI-EcoRI fragment (position 31-1724) labelled byrandom priming (19) with [α-³²P]dCTP (3000 Ci/mmol; Amersham) and havinga specific activity of 1.2×10⁹ cpm/μg. The final washes of the filtersare performed at 65° C. in 0.1×SSC/0.1% SDS solution.

Abbreviations used:

ChAT: choline acetyltransferase

ACh: acetylcholine

VMAT: vesicular monoamine transporter

TM: transmembrane domain

rVAT: rat vesicular acetylcholine transporter

SLIC: ligation of single-stranded cDNA

12 3925 base pairs nucleic acid single linear cDNA unknown 1 AAGCTTCCAAGCCACTTGTG AGCCCACTCA GGGTTTGGAG GCGGACGGGG TGGGGGTGGG 60 GTGGGGAAATGCAGAAAAGT GGGCGGAGGC TCTCAAGAGC CTAGGGAGGA TAAGGTCTGG 120 AAAGAAGAGGACCTGGGAGG AGTTAGATTG AGGAGTGGGA GAGTAGAAGG GGAGGGAAAT 180 AGGCGGGGCTGGAGGCCGGG GAGACCCGCG CACCGAAAAG CCCAAAGGGA GAGTCCAGGC 240 AGGGGGAGGTCAAAAAGGGT TAGCGTTGAC ATCCAGGACC CTGGGTGCAG AGAAAGACTC 300 CTCCTCTCAGTCCTCATACC CTCATAGTTC AGAATTAGCT GCCAAGACTT TCTGCCTAAG 360 GGCGGTGGGTCGGAACCAGA GCCTGAAGGC TCTGTACCTC CCCCCTCCCT TCCCGGAGGA 420 GGGGATGGGACGGGCTGGGG GCGGGATTGA GGAAGGGGGT TGCGTGCGCT GTGCCTCTGG 480 GTCCGGGCTGCGCGTTCCAG CTGCGAGAAC AGATGGAGGC AGCGCGGCTC ACCTCGGGGG 540 CTCCGTGCCCGCTGTGCGCC GAAGTCCAGG CTGAGGAGGA GGTCTAGAGC CCCCGGCTCT 600 CCCGTCTCCCACCAGGCTGC GGGGAACTGG CTGCCGCACC CCTCCTCCAA GTGGGGGTAG 660 AACGGAGTCTCACCCCCATA GGTCCCAGAA CTAAGGGGAA CATAGGGCCG GTTCCTCCCA 720 CTGCTCAGCCATCCCCAGGG GCTTGTCTAG GACTATAGCT CTCCAAATCC CCCTTCCCCT 780 GGCTTCCATCCTGGGCGCAT CTCAGAAGCG GACCCCTGCC CGGACGCGCC CCGCCCCCGG 840 CCCCCGCCCCGACGACGTCC TATTAGCATG AGCGACGCCA GTGGCCGGGG CACCACTCGG 900 GGGCCGAGACTCACCGCGTC ATAGCCCCAA GTGGAGGGAG AAAGAAAAAA AAAGAGGCGG 960 CGGTGGAGGAAGAGGCAAGA GCGGACGGGC GGGGAGGGCT GGAGAGACGG CGGGCGGCGG 1020 CAGCATGCCCCTGGGCGGGT GCACACGGCC TCTCTGCACC GCAGGGGCTG CTTCTGCTCT 1080 CTCTGGGCACCACGCGTCCA GTCTCCCGCC TCAGCCCCTC GGCTTGCCGG CCTTTGCGGT 1140 TGCGCTCGAAACATCGTCCA CTGGTCCCCG AAGCATCTAA GAGCAGCGGC GCCGCGCGGG 1200 ACAATCCTTGCTTTTTTCTG AGCTCGGGGA TATGAGCCCC ACAGCCACCT GAAGCGCAGG 1260 GGGCGCTACGGCTAGGACCG CGCCCCCGAA GTACCTTATC CTAGCCTCTG CACTGCGGGA 1320 CGCCGACACCCGACTCCGGT GGAGGCATCT TAGGGAAAGC AGCCGGTAGG GGCATGGAAC 1380 CCACCGCGCCAACCGGTCAG GCCCGGGCGG CGGCCACCAA ACTGTCGGAA GCGGTGGGAG 1440 CCGCGCTACAAGAGCCCCAG AGGCAGCGGC GCCTGGTGCT GGTCATCGTG TGCGTTGCAC 1500 TGTTACTGGACAACATGTTG TACATGGTCA TCGTGCCCAT TGTTCCCGAC TATATCGCCC 1560 ACATGCGCGGGGGCAGCGAG GGCCCGACCC TGGTCTCTGA GGTGTGGGAA CCCACTCTGC 1620 CGCCGCCCACTCTGGCTAAT GCCAGTGCCT ACTTGGCCAA CACGTCGGCG TCCCCGACGG 1680 CTGCCGGGTCGGCTCGGTCA ATCCTGCGAC CTCGCTACCC CACAGAAAGC GAAGATGTGA 1740 AGATAGGTGTGCTGTTTGCC TCCAAGGCTA TCCTGCAGCT TCTGGTGAAC CCCTTAAGCG 1800 GGCCTTTCATTGATCGCATG AGCTACGACG TGCCGCTGCT TATAGGCCTG GGCGTCATGT 1860 TCGCCTCCACAGTCATGTTT GCCTTTGCAG AAGACTATGC CACGCTCTTC GCTGCGCGCA 1920 GTCTACAAGGCCTGGGCTCG GCCTTCGCGG ACACGTCTGG CATTGCCATG ATCGCCGACA 1980 AGTATCCCGAGGAGCCTGAG CGCAGTCGTG CCCTGGGCGT GGCGCTAGCC TTTATTAGCT 2040 TTGGAAGCCTAGTGGCGCCA CCGTTTGGGG GCATCCTCTA CGAGTTCGCG GGCAAGCGTG 2100 TACCCTTTCTAGTGCTCGCC GCTGTGTCCC TTTTCGACGC GCTCCTGCTC CTGGCGGTGG 2160 CTAAGCCCTTCTCGGCTGCG GCTCGGGCGC GAGCCAACCT GCCGGTGGGC ACACCTATCC 2220 ATCGCCTCATGCTAGACCCT TACATCGCTG TGGTAGCCGG CGCGCTCACC ACTTGTAACA 2280 TTCCCCTTGCGTTCCTCGAG CCCACCATAG CCACGTGGAT GAAGCACACA ATGGCCGCAT 2340 CCGAGTGGGAGATGGGCATG GTTTGGCTGC CGGCTTTCGT GCCACACGTG TTAGGCGTCT 2400 ACCTCACCGTGCGCCTGGCG GCGCGTTATC CACACCTGCA GTGGCTGTAC GGCGCTCTCG 2460 GGCTAGCGGTAATTGGAGTG AGCTCTTGCG TCGTACCTGC CTGTCGCTCA TTCGCGCCGT 2520 TAGTGGTCTCGCTCTGCGGA CTCTGCTTCG GCATCGCGTT AGTGGACACA GCGCTCCTAC 2580 CCACGCTCGCCTTTCTGGTG GACGTGCGCC ACGTATCCGT CTATGGCAGT GTCTATGCCA 2640 TAGCTGACATCTCCTATTCT GTGGCCTACG CGCTCGGGCC CATAGTGGCA GGCCACATCG 2700 TTCACTCTCTTGGCTTTGAG CAGCTCAGCC TGGGCATGGG CCTGGCCAAC CTGCTCTACG 2760 CACCAGTCCTTCTTCTTTTG CGCAATGTAG GCCTCCTTAC ACGCTCGCGT TCGGAGCGCG 2820 ATGTGTTGCTTGATGAACCG CCGCAGGGTC TGTACGACGC GGTGCGCCTG CGTGAGGTGC 2880 AGGGCAAGGATGGCGGCGAA CCTTGTAGCC CACCTGGCCC TTTTGACGGG TGCGAGGACG 2940 ACTACAACTATTACTCCCGC AGCTAGCAGA CCCGCTTCTC CTCCAGGCCA CCTACCCGCC 3000 CCATTTAGGTCAAGATGGTC ATTCTGCAAG AGCACTGTCC AACTTTGGCT TGGGGCCCAC 3060 CTCCTCTAATGAATACCCTA GCCCCTCGCC CGTCCTGAAT TCCTTTGCTG GAATCCCTTC 3120 TCCATGACCCCTCCCAGTCT AGGCCCCTCC CAAACACACT CGTATTCATT GGGGAAATGG 3180 AGCAGGGAGGCAGAAGAAGC TGTTGGGCTC TTGGCAGAGG TGAAGAGGTG TGCGGGTGAT 3240 CGCCAATCACCTACTGAGAG CCCCCAAATA GAGTCATGCA TCTGTTTGTC CTTCCTGCGG 3300 ATCTTTCCAGTGCCAAACTT GGTCTCTGCA CTCCGGTGCC TCCGGCCTGA ATTAATAAAC 3360 CATATCTATCTGAGGAGGCC GAGTCTCTTT ACTGATGAGG GGTGGGTGGT GTGACACAAG 3420 ACCTAAGCACAGAGAAGGCT GCCTGGGTTT CACAGGTTCA GTCCAGACCT GAGGAGGAGG 3480 GGAAGCCTGAAGCGTCTTTG CTGCCTGGTA AAAGAACCCA AAGGAGGGCT CTCCCCCATG 3540 GATATTCAGAACACACACAC ACACACACAC ACTCACACAC ACACACACAC ACACACACAC 3600 ACACACGAACGATAGACAGA CAGACAGACA GACAGACAGA CAGACAGTCT CTCCCTTCCA 3660 AGTCCAGTGTAGCACCTGGA GGTTCCACCC GAGGGAGCCT GAGGATCTGC CTGGCCTTGG 3720 AGGATAGCTGGCACCAGGAA TTTTGGGTGC CAGGACTGGG CTTTCCTACA CAGTGGGAAC 3780 TCGCTTCATGTTGTCAAGAA AGGGAGCTGT TTTCTGCAGA GAAGAGGAGG TAGTCCCGTC 3840 TTTTAGGGTCCTGGCCTGGG GACAGTGTTC ATTAAGGATT CAGGCTCTTT CTGTGAAGAC 3900 TGAGAGGACACTTACCTGTG GATCC 3925 1593 base pairs nucleic acid single linear cDNAunknown CDS 1..1593 2 ATG GAA CCC ACC GCG CCA ACC GGT CAG GCC CGG GCGGCG GCC ACC AAA 48 Met Glu Pro Thr Ala Pro Thr Gly Gln Ala Arg Ala AlaAla Thr Lys 1 5 10 15 CTG TCG GAA GCG GTG GGA GCC GCG CTA CAA GAG CCCCAG AGG CAG CGG 96 Leu Ser Glu Ala Val Gly Ala Ala Leu Gln Glu Pro GlnArg Gln Arg 20 25 30 CGC CTG GTG CTG GTC ATC GTG TGC GTT GCA CTG TTA CTGGAC AAC ATG 144 Arg Leu Val Leu Val Ile Val Cys Val Ala Leu Leu Leu AspAsn Met 35 40 45 TTG TAC ATG GTC ATC GTG CCC ATT GTT CCC GAC TAT ATC GCCCAC ATG 192 Leu Tyr Met Val Ile Val Pro Ile Val Pro Asp Tyr Ile Ala HisMet 50 55 60 CGC GGG GGC AGC GAG GGC CCG ACC CTG GTC TCT GAG GTG TGG GAACCC 240 Arg Gly Gly Ser Glu Gly Pro Thr Leu Val Ser Glu Val Trp Glu Pro65 70 75 80 ACT CTG CCG CCG CCC ACT CTG GCT AAT GCC AGT GCC TAC TTG GCCAAC 288 Thr Leu Pro Pro Pro Thr Leu Ala Asn Ala Ser Ala Tyr Leu Ala Asn85 90 95 ACG TCG GCG TCC CCG ACG GCT GCC GGG TCG GCT CGG TCA ATC CTG CGA336 Thr Ser Ala Ser Pro Thr Ala Ala Gly Ser Ala Arg Ser Ile Leu Arg 100105 110 CCT CGC TAC CCC ACA GAA AGC GAA GAT GTG AAG ATA GGT GTG CTG TTT384 Pro Arg Tyr Pro Thr Glu Ser Glu Asp Val Lys Ile Gly Val Leu Phe 115120 125 GCC TCC AAG GCT ATC CTG CAG CTT CTG GTG AAC CCC TTA AGC GGG CCT432 Ala Ser Lys Ala Ile Leu Gln Leu Leu Val Asn Pro Leu Ser Gly Pro 130135 140 TTC ATT GAT CGC ATG AGC TAC GAC GTG CCG CTG CTT ATA GGC CTG GGC480 Phe Ile Asp Arg Met Ser Tyr Asp Val Pro Leu Leu Ile Gly Leu Gly 145150 155 160 GTC ATG TTC GCC TCC ACA GTC ATG TTT GCC TTT GCA GAA GAC TATGCC 528 Val Met Phe Ala Ser Thr Val Met Phe Ala Phe Ala Glu Asp Tyr Ala165 170 175 ACG CTC TTC GCT GCG CGC AGT CTA CAA GGC CTG GGC TCG GCC TTCGCG 576 Thr Leu Phe Ala Ala Arg Ser Leu Gln Gly Leu Gly Ser Ala Phe Ala180 185 190 GAC ACG TCT GGC ATT GCC ATG ATC GCC GAC AAG TAT CCC GAG GAGCCT 624 Asp Thr Ser Gly Ile Ala Met Ile Ala Asp Lys Tyr Pro Glu Glu Pro195 200 205 GAG CGC AGT CGT GCC CTG GGC GTG GCG CTA GCC TTT ATT AGC TTTGGA 672 Glu Arg Ser Arg Ala Leu Gly Val Ala Leu Ala Phe Ile Ser Phe Gly210 215 220 AGC CTA GTG GCG CCA CCG TTT GGG GGC ATC CTC TAC GAG TTC GCGGGC 720 Ser Leu Val Ala Pro Pro Phe Gly Gly Ile Leu Tyr Glu Phe Ala Gly225 230 235 240 AAG CGT GTA CCC TTT CTA GTG CTC GCC GCT GTG TCC CTT TTCGAC GCG 768 Lys Arg Val Pro Phe Leu Val Leu Ala Ala Val Ser Leu Phe AspAla 245 250 255 CTC CTG CTC CTG GCG GTG GCT AAG CCC TTC TCG GCT GCG GCTCGG GCG 816 Leu Leu Leu Leu Ala Val Ala Lys Pro Phe Ser Ala Ala Ala ArgAla 260 265 270 CGA GCC AAC CTG CCG GTG GGC ACA CCT ATC CAT CGC CTC ATGCTA GAC 864 Arg Ala Asn Leu Pro Val Gly Thr Pro Ile His Arg Leu Met LeuAsp 275 280 285 CCT TAC ATC GCT GTG GTA GCC GGC GCG CTC ACC ACT TGT AACATT CCC 912 Pro Tyr Ile Ala Val Val Ala Gly Ala Leu Thr Thr Cys Asn IlePro 290 295 300 CTT GCG TTC CTC GAG CCC ACC ATA GCC ACG TGG ATG AAG CACACA ATG 960 Leu Ala Phe Leu Glu Pro Thr Ile Ala Thr Trp Met Lys His ThrMet 305 310 315 320 GCC GCA TCC GAG TGG GAG ATG GGC ATG GTT TGG CTG CCGGCT TTC GTG 1008 Ala Ala Ser Glu Trp Glu Met Gly Met Val Trp Leu Pro AlaPhe Val 325 330 335 CCA CAC GTG TTA GGC GTC TAC CTC ACC GTG CGC CTG GCGGCG CGT TAT 1056 Pro His Val Leu Gly Val Tyr Leu Thr Val Arg Leu Ala AlaArg Tyr 340 345 350 CCA CAC CTG CAG TGG CTG TAC GGC GCT CTC GGG CTA GCGGTA ATT GGA 1104 Pro His Leu Gln Trp Leu Tyr Gly Ala Leu Gly Leu Ala ValIle Gly 355 360 365 GTG AGC TCT TGC GTC GTA CCT GCC TGT CGC TCA TTC GCGCCG TTA GTG 1152 Val Ser Ser Cys Val Val Pro Ala Cys Arg Ser Phe Ala ProLeu Val 370 375 380 GTC TCG CTC TGC GGA CTC TGC TTC GGC ATC GCG TTA GTGGAC ACA GCG 1200 Val Ser Leu Cys Gly Leu Cys Phe Gly Ile Ala Leu Val AspThr Ala 385 390 395 400 CTC CTA CCC ACG CTC GCC TTT CTG GTG GAC GTG CGCCAC GTA TCC GTC 1248 Leu Leu Pro Thr Leu Ala Phe Leu Val Asp Val Arg HisVal Ser Val 405 410 415 TAT GGC AGT GTC TAT GCC ATA GCT GAC ATC TCC TATTCT GTG GCC TAC 1296 Tyr Gly Ser Val Tyr Ala Ile Ala Asp Ile Ser Tyr SerVal Ala Tyr 420 425 430 GCG CTC GGG CCC ATA GTG GCA GGC CAC ATC GTT CACTCT CTT GGC TTT 1344 Ala Leu Gly Pro Ile Val Ala Gly His Ile Val His SerLeu Gly Phe 435 440 445 GAG CAG CTC AGC CTG GGC ATG GGC CTG GCC AAC CTGCTC TAC GCA CCA 1392 Glu Gln Leu Ser Leu Gly Met Gly Leu Ala Asn Leu LeuTyr Ala Pro 450 455 460 GTC CTT CTT CTT TTG CGC AAT GTA GGC CTC CTT ACACGC TCG CGT TCG 1440 Val Leu Leu Leu Leu Arg Asn Val Gly Leu Leu Thr ArgSer Arg Ser 465 470 475 480 GAG CGC GAT GTG TTG CTT GAT GAA CCG CCG CAGGGT CTG TAC GAC GCG 1488 Glu Arg Asp Val Leu Leu Asp Glu Pro Pro Gln GlyLeu Tyr Asp Ala 485 490 495 GTG CGC CTG CGT GAG GTG CAG GGC AAG GAT GGCGGC GAA CCT TGT AGC 1536 Val Arg Leu Arg Glu Val Gln Gly Lys Asp Gly GlyGlu Pro Cys Ser 500 505 510 CCA CCT GGC CCT TTT GAC GGG TGC GAG GAC GACTAC AAC TAT TAC TCC 1584 Pro Pro Gly Pro Phe Asp Gly Cys Glu Asp Asp TyrAsn Tyr Tyr Ser 515 520 525 CGC AGC TAG 1593 Arg Ser * 530 530 aminoacids amino acid linear protein unknown 3 Met Glu Pro Thr Ala Pro ThrGly Gln Ala Arg Ala Ala Ala Thr Lys 1 5 10 15 Leu Ser Glu Ala Val GlyAla Ala Leu Gln Glu Pro Gln Arg Gln Arg 20 25 30 Arg Leu Val Leu Val IleVal Cys Val Ala Leu Leu Leu Asp Asn Met 35 40 45 Leu Tyr Met Val Ile ValPro Ile Val Pro Asp Tyr Ile Ala His Met 50 55 60 Arg Gly Gly Ser Glu GlyPro Thr Leu Val Ser Glu Val Trp Glu Pro 65 70 75 80 Thr Leu Pro Pro ProThr Leu Ala Asn Ala Ser Ala Tyr Leu Ala Asn 85 90 95 Thr Ser Ala Ser ProThr Ala Ala Gly Ser Ala Arg Ser Ile Leu Arg 100 105 110 Pro Arg Tyr ProThr Glu Ser Glu Asp Val Lys Ile Gly Val Leu Phe 115 120 125 Ala Ser LysAla Ile Leu Gln Leu Leu Val Asn Pro Leu Ser Gly Pro 130 135 140 Phe IleAsp Arg Met Ser Tyr Asp Val Pro Leu Leu Ile Gly Leu Gly 145 150 155 160Val Met Phe Ala Ser Thr Val Met Phe Ala Phe Ala Glu Asp Tyr Ala 165 170175 Thr Leu Phe Ala Ala Arg Ser Leu Gln Gly Leu Gly Ser Ala Phe Ala 180185 190 Asp Thr Ser Gly Ile Ala Met Ile Ala Asp Lys Tyr Pro Glu Glu Pro195 200 205 Glu Arg Ser Arg Ala Leu Gly Val Ala Leu Ala Phe Ile Ser PheGly 210 215 220 Ser Leu Val Ala Pro Pro Phe Gly Gly Ile Leu Tyr Glu PheAla Gly 225 230 235 240 Lys Arg Val Pro Phe Leu Val Leu Ala Ala Val SerLeu Phe Asp Ala 245 250 255 Leu Leu Leu Leu Ala Val Ala Lys Pro Phe SerAla Ala Ala Arg Ala 260 265 270 Arg Ala Asn Leu Pro Val Gly Thr Pro IleHis Arg Leu Met Leu Asp 275 280 285 Pro Tyr Ile Ala Val Val Ala Gly AlaLeu Thr Thr Cys Asn Ile Pro 290 295 300 Leu Ala Phe Leu Glu Pro Thr IleAla Thr Trp Met Lys His Thr Met 305 310 315 320 Ala Ala Ser Glu Trp GluMet Gly Met Val Trp Leu Pro Ala Phe Val 325 330 335 Pro His Val Leu GlyVal Tyr Leu Thr Val Arg Leu Ala Ala Arg Tyr 340 345 350 Pro His Leu GlnTrp Leu Tyr Gly Ala Leu Gly Leu Ala Val Ile Gly 355 360 365 Val Ser SerCys Val Val Pro Ala Cys Arg Ser Phe Ala Pro Leu Val 370 375 380 Val SerLeu Cys Gly Leu Cys Phe Gly Ile Ala Leu Val Asp Thr Ala 385 390 395 400Leu Leu Pro Thr Leu Ala Phe Leu Val Asp Val Arg His Val Ser Val 405 410415 Tyr Gly Ser Val Tyr Ala Ile Ala Asp Ile Ser Tyr Ser Val Ala Tyr 420425 430 Ala Leu Gly Pro Ile Val Ala Gly His Ile Val His Ser Leu Gly Phe435 440 445 Glu Gln Leu Ser Leu Gly Met Gly Leu Ala Asn Leu Leu Tyr AlaPro 450 455 460 Val Leu Leu Leu Leu Arg Asn Val Gly Leu Leu Thr Arg SerArg Ser 465 470 475 480 Glu Arg Asp Val Leu Leu Asp Glu Pro Pro Gln GlyLeu Tyr Asp Ala 485 490 495 Val Arg Leu Arg Glu Val Gln Gly Lys Asp GlyGly Glu Pro Cys Ser 500 505 510 Pro Pro Gly Pro Phe Asp Gly Cys Glu AspAsp Tyr Asn Tyr Tyr Ser 515 520 525 Arg Ser 530 1902 base pairs nucleicacid single linear cDNA unknown 4 AAGCTTTTTG CTGAAGGCGA TCCTTCCCTGACTGTGGCCC CATATCCTTC CGATGTAAAG 60 TCTCTGCCTT CTTATTTGGA AGCTAAAGAATGTCATTTAG GGACCTGGAA GGAGCCAAAC 120 CCATGCCTCC CTCACTCCGA TTTTATTTGCCTAGGTGTGT GTGCCACACT GCACATGGAG 180 TGACACAGGC GTAATGAAAC ACAAGGGAAAGGGAGGTGGC ACGAAGACCT CAAAGCAGGT 240 CCCAACAGCC TGGAAACTCA ACAAGTGACCATCTCCCTTC TCCATGCCTA GTTTCCTATG 300 GGGGTGTGTG ATGGGGGTGG GGTGGATAGCATCTACATTG TACCAAGATT GAGGAAGGAC 360 GGAGTGAGCT CACCCATGGC CCTGGCACGAAACCAGTGGG TCTGCACCTT ATCAGAGAGG 420 AACGGAGGAG CAGAGAGTTG GGTGCGAGTCCCAGAATGAA TTGGCTTCAT TCTCAGGAAG 480 CCATGGATTA TTATGTGGGC TGGAGGGCTGAGAGACAAGG TCTGCCTCTT GCTAGCACTG 540 GGAAGTTTCT TTCCTGAAAG ATTCTACTGTCCACTAACCA CCAGAAAGAA AACTGCTGCC 600 TGCGGTGTTG TCCTGCACCC TGATTTTATTCAGTCAGAGC GGCAATATAT GAATAAGTAA 660 TCACGACCAT GTAGGGCAGG AAATACCTCTTTAAAAACCA CCAGTGGGAT TTTCATAGAA 720 AAGTCAGAGG AACCCAGAGC GTTCTTAAACTCCAGTGTCT GGAGAGCCAA GCAGGTGACA 780 GACAGAGTTC ATGAAGCAAG CTGAGGGAAAACCCCATCGC TGGAGTGTGT GCTATGAACA 840 AAGGCACCAG GATAATATTT AAAGTTGTTTCACCGCACTT TCTGCAAGGT GGATCTTGGT 900 AATCTCTTGA TGAACAGAGG CCAAGTGGGCCAGGGTTAGT GGGGAATGTG GCTTGCCCAA 960 TGCTTTCCAT AGCCATGGCT GTCCCCATAGTTAGGCTTCC TCCCTCATTT TGAGGGCAGC 1020 TGGGCCACTG CAGGGTATGT GATGGGCAGCTCTCTACTCA GATGAGTCTG TTCCTTTCAG 1080 GAGTACCTGT GTACAGGTTG GGAGGTTGGGCACTAGACCA CTTGATTGCT GCCTCTCTCC 1140 CTGACTCTGT TCTCCATCAC CTCCTCTTGCAACTGGCTTA AGAACAGAAG CCAGCCAATT 1200 TTCCAGCTCC TTCTGTATCT TCCACCATCCCAGACAGAGT CCAGGCTCAC AATGCCTACC 1260 CAACTGAGGA AGAAATCAGA GAGTCAGGATGCTCCCGGTG TCTGACTGCC CTTCACAAGA 1320 CCCTCATGAA CACAAGGCAG CAAGCACATGCTATAACAAC AACGGCAAAT GCTAATGATT 1380 CACACCACGC GTGTGCCACA CCCTAGTTGTACGTACTCCT ATTCCATTTT ACAGATAAAT 1440 AAAGGGGCGG TGGGGGCAGA GGGAGGAAACAACGGCTCCC TTGGCACTGT TATCTAGTAA 1500 GTGGCAGCAC TGGGAGCCAT CCTATCTGTCTGCATCTGGA GCTCAAATCG TGATGCCTCC 1560 TTCGGTGGAG GAAGGCTAGC TTGGGATATGGAGGCTACTG TGACTCCGGA AGACAGAGAA 1620 AAGTCCAATC TCAACAACGT CACCACTATCCCCAATCTCA GCTGACTGGC ATCCTCTCTC 1680 CTGCCAGTCT GTGGACGGGA ACGCGGGCCTCACGTGCCAT CTAGGGTCAA AACTCGTCTG 1740 AGGACACACA CTGGGCCCAA CGCAGAGGCTGATCTGTTCA GCCTGTCGGC TGCAAGCCAG 1800 GACTCTCAGC TTGTGCAGCA CCCCCGGAAGGAAGGTGAGC CTTCCTAAGC CTCTACTGAC 1860 AGCAAAGCTG CAGAGGCCCT GCCGCGTGAGACCCAGAAGC TT 1902 6 base pairs nucleic acid single linear other nucleicacid unknown 5 AATAAA 6 23 base pairs nucleic acid single linear othernucleic acid unknown 6 CTGTCGCTGC AAGCCAGGAC TCT 23 22 base pairsnucleic acid single linear other nucleic acid unknown 7 TGGAGGAAGAGGCAAGAGCG GA 22 24 base pairs nucleic acid single linear other nucleicacid unknown 8 ACCGGTTGGC GCGGTGGGTT CCAT 24 22 base pairs nucleic acidsingle linear other nucleic acid unknown 9 CACCGCTTCC GACAGTTTGG TG 2224 base pairs nucleic acid single linear other nucleic acid unknown 10TGGGCGATAT AGTCGGGAAC AATG 24 50 base pairs nucleic acid single linearother nucleic acid unknown 11 CTGCATCTAT CTAATGCTCC TCTCGCTACCTGCTCACTCT GCGTGACATC 50 25 base pairs nucleic acid single linear othernucleic acid unknown 12 GATGTCACGC AGAGTGAGCA GGTAG 25

What is claimed is:
 1. A recombinant viral vector comprising a nucleicacid sequence encoding a rat vesicular acetylcholine transport protein,wherein said nucleic acid sequence comprises a sequence as set forth inSEQ ID NO:
 1. 2. The recombinant viral vector according to claim 1,wherein said vector is derived from a virus selected from the groupconsisting of adenovirus, retrovirus, adeno-associated virus,herpesvirus, cytomegalovirus, and vaccinia virus.
 3. The recombinantviral vector according to claim 1, wherein said viral vector isreplication defective.
 4. A composition comprising one or more vectorsaccording to claim
 1. 5. A method for expressing a vesicularacetylcholine transport protein in a cultured cell comprisingadministration to the cell of the recombinant viral vector according toclaim 1 and culturing the cell under conditions that result in theexpression of the vesicular acetylcholine transport protein.
 6. A methodfor directing expression of a gene encoding a rat vesicularacetylcholine transport protein in a cultured cell comprising 1)operably linking said gene to a promoter region selected from the groupconsisting of a region lying between positions 2 and 583 of Seq ID No:1, and a promoter region comprising SEQ ID No: 4; 2) administering thegene operably linked to a promoter region as formed in step 1) to thecell; and 3) culturing the cell under conditions that result in theexpression of the vesicular acetylcholine transport protein.